Inhibition of PKC to treat permability failure

ABSTRACT

The invention features a method of treating a subject, e.g., a subject having permeability disjunction, which includes administering to the subject peritoneal dialysis fluid which includes an inhibitor of PKC, e.g., an inhibitor of PKC β. The invention also features an improved peritoneal dialysis fluid and methods of making such dialysis fluid.

This application is a continuation of, and claims the benefit of, U.S.patent application Ser. No. 09/524,459, filed Mar. 10, 2000, whichclaims the benefit of Provisional Application No. 60/124,043, filed Mar.12, 1999, the contents of which are incorporated in their entirety.

BACKGROUND OF THE INVENTION

The invention relates to peritoneal dialysis fluids, and methods ofmaking and using them. More particularly, it relates to the methods forpreventing and treating complications due to peritoneal dialysis.

Peritoneal dialysis can be used to treat patients having chronic renalfailure. In this procedure, peritoneal dialysis fluid is introduced intothe peritoneal cavity of a subject. The fluid generally has a highglucose level, often in the 100 mM range. Introduction of thisrelatively high molarity fluid into the peritoneum causes the osmoticextraction of toxins, for example, urea creatinine, and other substancesnormally removed by the kidney, from the blood. It can also reduce thelevel of fluid in the patient.

SUMMARY OF THE INVENTION

The invention is based, in part, on the discovery that the use ofperitoneal dialysis fluid, especially prolonged use, can result indecreasing permeability between the peritoneal dialysis fluid and theblood compartment, that high levels of glucose are involved in changesin the cells lining the peritoneum, that the high glucose levelsactivate PKC, and that the inhibition of PKC can prevent this chroniccomplication. The invention can treat an undesirable decrease in theefficiency of exchange between the peritoneal dialysis fluid and thevascular compartment.

Accordingly, the invention features, a method of treating a subject. Themethod includes:

introducing peritoneal dialysis fluid into the peritoneum of thesubject; and

inhibiting a PKC in the subject, thereby treating said subject.

In a preferred embodiment, an inhibitor of PKC is included in theperitoneal dialysis fluid. The inhibitor is preferably a specificinhibitor of PKC. The inhibitor can be an inhibitor of a PKC β, e.g., β1or β2, γ, δ, or other isoform. The inhibitor can be, for example, a bis(indolyl) maleimide, for example, the PKC β inhibitor LY333531, which isdescribed in Ishi et al. (1996) Science 272:728-731, hereby incorporatedby reference. LY333531 can be present in the dialysis fluid at about1-1,000, 5-750, 20-500, but more preferably 50-500 nanometers.

In a preferred embodiment, the concentration of glucose in theperitoneal dialysis fluid is 200 nM.

Peritoneal dialysis fluid of the invention is particularly useful forsubjects who are already peritoneal dialysis patients. In preferredembodiments, the subject has been a peritoneal dialysis patient for atleast 2, 4, 6, 12, or 24 months, e.g., the subject has been administeredperitoneal dialysis fluid, either one with or without a PKC inhibitor,periodically for at least 2, 4, 6, 12, or 24 months, or it has been atleast 2, 4, 6, 12, or 24 months since the subjects first peritonealdialysis administration. Peritoneal dialysis fluid of the invention canbe administered to patients who have already developed permeabilitydisjunction.

In other embodiments peritoneal dialysis fluid of the invention isuseful for subjects who have not yet had peritoneal dialysis. Peritonealdialysis fluid of the invention can be administered to patients who havenot yet developed permeability disjunction.

In another aspect, the invention features, a method of treating asubject. The method includes:

introducing peritoneal dialysis fluid and an inhibitor of a PKC into thesubject, thereby treating the subject.

In a preferred embodiment, the peritoneal dialysis fluid and PKCinhibitor are co-administered to the subject; the peritoneal dialysisfluid and PKC inhibitor are introduced separately into the subject; theperitoneal dialysis fluid and PKC inhibitor are combined prior tointroduction into the subject and administered together.

In a preferred embodiment, an inhibitor of PKC is included in theperitoneal dialysis fluid. The inhibitor is preferably a specificinhibitor of PKC. The inhibitor can be an inhibitor of a PKC β, e.g., β1or β2, γ, δ, or other isoform, or combinations thereof. The inhibitorcan be, for example, a bis (indolyl) maleimide, for example, the PKC βinhibitor LY333531. LY333531 can be present in the dialysis fluid atabout 1-1,000, 5-750, 20-500, but more preferably 50-500 nanometers.

In a preferred embodiment, the concentration of glucose in theperitoneal dialysis fluid is 200 nM.

In a preferred embodiment, the subject is administered a second, third,fourth, or fifth, infusion of peritoneal dialysis fluid.

Peritoneal dialysis fluid of the invention is particularly useful forsubjects who are already peritoneal dialysis patients. In preferredembodiments the subject has been a peritoneal dialysis patient for atleast 2, 4, 6, 12, or 24 months, e.g., the subject has been administeredperitoneal dialysis fluid, either one with or without a PKC inhibitor,periodically for at least 2, 4, 6, 12, or 24 months, or it has been atleast 2, 4, 6, 12, or 24 months since the subjects first peritonealdialysis administration. Peritoneal dialysis fluid of the invention canbe administered to patients which have already developed permeabilitydisjunction.

In other embodiments, peritoneal dialysis fluid of the invention isuseful for subjects who have not yet had peritoneal dialysis. Peritonealdialysis fluid of the invention can be administered to patients whichhave not yet developed permeability disjunction.

In a preferred embodiment, the subject is at risk for renal failure, forexample, the subject is a patient in end-stage renal failure.

In another aspect, the invention features, a peritoneal dialysis fluidwhich includes an inhibitor of a PKC.

In a preferred embodiment, the peritoneal dialysis fluid is onedescribed herein.

In a preferred embodiment, an inhibitor of PKC is included in theperitoneal dialysis fluid. The inhibitor is preferably a specificinhibitor of PKC. The inhibitor can be an inhibitor of a PKC β, e.g., β1or β2, γ, δ, or other isoform, or combinations thereof. The inhibitorcan be, for example, a bis (indolyl) maleimide, for example, the PKC βinhibitor LY333531. LY333531 can be present in the dialysis fluid atabout 1-1,000, 5-750, 20-500, but more preferably 50-500 nM.

In a preferred embodiment, the concentration of glucose in theperitoneal dialysis fluid is 200 nM.

In yet another aspect, the invention features, a method of making animproved peritoneal dialysis fluid. The method includes, providing aperitoneal dialysis fluid and adding to that fluid an inhibitor of aPKC, for example an inhibitor described herein, for example LY333531.

In a preferred embodiment, an inhibitor of PKC is included in theperitoneal dialysis fluid. The inhibitor is preferably a specificinhibitor of PKC. The inhibitor can be an inhibitor of a PKC β, e.g., β1or β2, γ, δ, or other isoform, or combinations thereof. The inhibitorcan be, for example, a bis (indolyl) maleimide, for example, the PKC βinhibitor LY333531. LY333531 can be present in the dialysis fluid atabout 1-1,000, 5-750, 20-500, but more preferably 50-500 nM.

In a preferred embodiment, the concentration of glucose in theperitoneal dialysis fluid is 200 nM.

Subject, as used herein, can refer to a human subject, or a non-humananimal, for example, a horse, cow, goat, pig, sheep or other veterinary,food or fiber producing animal, in need of dialysis. The subject can bean individual at risk for (e.g., the individual can have or bepredisposed to have) end-stage renal disease, from any cause.

Other features and advantages of the invention will be apparent from thedescription herein and from the claims.

DETAILED DESCRIPTION OF THE DRAWINGS

FIG. 1 shows PKC activity of primary human mesothelial cells at variousglucose levels (i.e., low glucose, 60 mM or 150 mM glucose). The PKCactivity was determined in the presence or absence or PMA, an activatorof PKC.

FIG. 2 shows the effect of treatment with vitamin E on PKC activity ofhuman primary mesothelial cells. The mesothelial cells were exposed tolow or high (150 mM) levels of glucose in the presence or absence ofPMA, an activator of PKC, and the presence or absence of vitamin E, aPKC inhibitor.

FIG. 3 shows PKC activity of primary human mesothelial cells cultured inlow glucose concentrations, high glucose concentrations (i.e., 60 mM or150 mM), or mannitol. The PKC activity was determined in the presence orabsence or PMA, an activator of PKC.

FIG. 4 shows DAG activity of transformed mesothelial cells at lowglucose levels (5.5 mM) and high glucose levels (60 mM).

DETAILED DESCRIPTION

Peritoneal dialysis for chronic renal failure is used by some patientswith renal failure. The fluid for peritoneal dialysis uses glucose at400-mM range. While not wishing to be bound by theory, it appears thatat this concentration level, glucose causes complications which can leadto decreasing permeability between the peritoneal dialysis fluid and theblood compartment. The elevation of high glucose causes changes in thecells lining the peritoneum. The invention is based, in part on, PKCactivation, which is believed to cause this chronic decrease in theexchange between the peritoneal fluid and a vascular compartment.

PKC activation induced by hyperglycemia either in the peritoneal fluidor in the blood causes cells to behave abnormally. The inhibition of PKCβ isoform by the inhibitor LY333531 can prevent many of the vascularchanges induced by glucose levels up to 20-30 mM. The higherconcentration of glucose as used in peritoneal dialysis will alsoincrease PCK activation. Inhibition of PKC activation using a PKC β,e.g., β1 or β2, γ, δ, or other isoform, or combinations thereof,inhibitor will prevent the thickening and the decrease in exchangebetween peritoneal and vascular compartments, which will improve theefficiency of peritoneal dialysis for chronic renal failure.

As shown in FIGS. 1-3, exposure of human mesothelial cells, which arethe cells that line the peritoneum, to glucose levels of 60 mM or 150 mMresults in an increase of PKC activity. These concentrations of glucoseare standard for dialysis. In addition, as shown in FIG. 2, exposure ofthese cells to vitamin E, which is an inhibitor of PKC, resulted in adecrease of PKC activity. It was also found that high glucose levels of60 mM or 150 mM increased DAG activity. These results demonstrate thatglucose concentrations used for peritoneal dialysis activate the DAG-PKCpathways. It has been reported that activation of these pathwaysincreases fibrosis. These results also show that inhibitors of PKC suchas vitamin E decrease PKC activity. Therefore, inhibition of PKC candecrease fibrosis and reduce or prevent permeability failure of theperitoneal membrane.

Other embodiments are within the following claim.

1. A method of treating a subject, comprising: introducing into saidsubject a peritoneal dialysis fluid which includes an inhibitor of aPKC, thereby treating said subject.
 2. The method of claim 1, whereinsaid inhibitor is a specific inhibitor of PKC.
 3. The method of claim 2,wherein said inhibitor is selected from the group consisting of: aninhibitor of a PKC β, an inhibitor of PKC γ, and an inhibitor of PKC δ.4. The method of claim 2, wherein said inhibitor is an inhibitor of PKCβ.
 5. The method of claim 4, wherein said inhibitor is an inhibitor ofPKC β1.
 6. The method of claim 4, wherein said inhibitor is a bis(indolyl) maleimide.
 7. The method of claim 6, wherein said inhibitor isLY333531.
 8. The method of claim 7, wherein said LY333531 is present insaid dialysis fluid at about 1-1,000 nanometers.
 9. The method of claim1, wherein said dialysis fluid has a concentration of glucose of about200 nM.
 10. The method of claim 1, wherein said subject has previouslyreceived peritoneal dialysis.
 11. The method of claim 1, wherein saidsubject has been a peritoneal dialysis patient for at least 2 to 24months.
 12. The method of claim 1, wherein said subject has alreadydeveloped permeability disjunction.
 13. The method of claim 1, whereinsaid subject has not yet developed permeability disjunction.
 14. Themethod of claim 1, wherein said subject is at risk for renal failure.15. The method of claim 14, wherein said subject is in end-stage renalfailure.
 16. A peritoneal dialysis fluid comprising an inhibitor of aPKC.
 17. The dialysis fluid of claim 16, wherein said inhibitor is aspecific inhibitor of PKC.
 18. The dialysis fluid of claim 17, whereinsaid inhibitor is an inhibitor of PKC β.
 19. The dialysis fluid of claim18, wherein said inhibitor is a bis (indolyl) maleimide.
 20. Thedialysis fluid of claim 19, wherein said inhibitor is LY333531.
 21. Thedialysis fluid of claim 20, wherein said LY333531 is present in saiddialysis fluid at about 1-1,000 nanometers.
 22. The dialysis fluid ofclaim 16, wherein said dialysis fluid has a concentration of glucose ofabout 200 nM.
 23. A method of making an improved peritoneal dialysisfluid, comprising: providing a peritoneal dialysis fluid; and adding tothat fluid an inhibitor of a PKC, to thereby provide an improveddialysis fluid.
 24. The method of claim 23, wherein said inhibitor isLY333531.